Single nucleotide detection method

ABSTRACT

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

This invention relates to a method for characterising polynucleotidessuch as those derived from naturally occurring RNA or DNA by capturingand detecting an ordered sequence of single nucleotide bases generatedtherefrom by progressive pyrophosphorolysis.

Next generation sequencing of genetic material is already making asignificant impact on the biological sciences in general and medicine inparticular as the unit cost of sequencing falls in line with the comingto market of faster and faster sequencing machines. Thus, in one suchmachine, a double-stranded DNA analyte is first broken down into aplurality of smaller polynucleotide fragments each of which is firstadenylated on both ends of one strand so that a single-stranded firstoligonucleotide can be bound to both ends of its compliment byhybridisation to the unpaired adenine base. The treated fragments soobtained are then size-selected and captured on a surface coated withbound single-stranded second oligonucleotides which themselves are thesequence compliment of the first so that in effect a library ofsurface-bound double-stranded fragments can be created by furtherhybridisation. In a subsequent clustering step, these library componentsare then clonally amplified millions of times on the surface usingextension and isothermal bridging reactions to utilise unused secondoligonucleotides. This, in effect, creates a dense concentration of thepolynucleotide fragment bound to the surface through one of its strands.The unbound complimentary strand of each fragment is then removed toleave bound single-stranded fragments ready for sequencing. In thesequencing stage, each of these single-stranded fragments is primed andits complimentary strand recreated by extension using the polymerasechain reaction and a mixture of the four characteristic nucleotide basesof DNA in dideoxynucleotide triphosphate (ddNTP) form. Each ddNTP typeis end-blocked with a moiety which is labelled with a differentfluorophore fluorescing at a different wavelength. The extensionreaction then takes the form of a cycle of three steps; first therelevant ddNTP is incorporated into to the growing strand; secondly thenucleotide base it contains is identified by illuminating the sample anddetecting the wavelength of the fluorescence and finally the end blockand its associated fluorophore are removed to allow the next extensionevent to occur. By this means, the sequence of the complimentary strandcan be built up base-by-base. It will be appreciated that, whilst thisapproach can be highly automated and can generate sequence reads of highaccuracy, its speed of operation is limited by the rate of the extensioncycle. Thus, in practice, use of the technology tends to involveparallel processing of relatively short polynucleotide fragments andassembly of the whole sequence from the various reads obtainedtherefrom. This in itself can lead to computational complexities and thepotential introduction of errors.

More recently efforts have been made to develop direct sequencingmethods. For example, WO 2009/030953 discloses a new fast sequencer inwhich inter alia the sequence of nucleotide bases or base pairs in asingle- or double-stranded polynucleotide sample (e.g. naturallyoccurring RNA or DNA) is read by translocating the same through anano-perforated substrate provided with plasmonic nanostructuresjuxtaposed within or adjacent the outlet of the nanopores. In thisdevice, the plasmonic nanostructures define detection windows(essentially an electromagnetic field) within which each nucleotide base(optionally labelled) is in turn induced to fluoresce or Raman scatterphotons in a characteristic way by interaction with incident light. Thephotons so generated are then detected remotely, multiplexed andconverted into a data stream whose information content is characteristicof the nucleotide base sequence associated with the polynucleotide. Thissequence can then be recovered from the data stream using computationalalgorithms embodied in corresponding software programmed into amicroprocessor integral therewith or in an ancillary computing deviceattached thereto. Further background on the use of plasmonicnanostructures and their associated resonance characteristics can befound in for example Adv. Mat. 2004, 16(19) pp. 1685-1706.

Another apparatus for fast sequencing polynucleotides is described, forexample, in U.S. Pat. No. 6,627,067, U.S. Pat. No. 6,267,872 and U.S.Pat. No. 6,746,594. In its simplest form, this device employselectrodes, instead of plasmonic nanostructures, to define the detectionwindow across the substrate or in or around the outlet of the nanopore.A potential difference is then applied across the electrodes and changesin the electrical characteristics of the ionic medium flowingtherebetween, as a consequence of the electrophoretic translocation ofthe polynucleotide and associated electrolyte through the nanopore, ismeasured as a function of time. In this device, as the variousindividual nucleotide bases pass through the detection window theycontinuously block and unblock it causing ‘events’ which give rise tocharacteristic fluctuations in current flow or resistivity. Thesefluctuations are then used to generate a suitable data stream foranalysis as described above.

The generation of stable droplet streams, especially microdropletstreams, is another developing area of technology that already hasapplications in molecular biology. For example, U.S. Pat. No. 7,708,949discloses a novel microfluidic method for generating stable waterdroplets in oil whilst for example US2011/0250597 describes utilisationof this technology to generate microdroplets containing a nucleic acidtemplate (typically a polynucleotide DNA or RNA fragment) and aplurality of primer pairs that enable the template to be amplified usingthe polymerase chain reaction. Other patent applications relating to thefield generally include JP2004/290977, JP2004/351417, US2012/0122714,US2011/0000560, US2010/01376163, US2010/0022414 and US2008/0003142.

WO 2004/002627 discloses a method for creating liquid-liquid andgas-liquid dispersions using various devices comprising creating adiscontinuous section between upstream and downstream microfluidicregions. However its application to single nucleotide DNA sequencing isnot taught.

WO 2010/077859 teaches a droplet actuator comprising a substrateprovided with electrodes, a reactor path and nucleotide base,wash-buffer, sample and enzyme reservoirs. Whist the actuator isgenerically said to be useful for the amplification and sequencing ofnucleic acids, there is no teaching of the analyte degradation method wedescribe below. Rather, it is concerned with a completely differentapproach; observing the synthesis of a complimentary strand of theanalyte using pyrosequencing. US 2009/0280475 is concerned with similarsubject-matter.

Biological probes, which typically comprise single-strandedoligonucleotides of known sequence order less than 1000 nucleotideslong, are widely used in analytical molecular biology. Such probestypically work by attaching themselves to the target (for example onederived from the DNA of a naturally-occurring pathogen) when sufficientsequence complimentarity exists between the nucleotide bases of theprobe and the target. Typically the nucleotides of such probes arelabelled with detectable elements such as radioactive or fluorescentmarkers so that when the probe is used to treat an analyte solution orsubstrate in or on which the target is thought to have been captured,the presence or absence of the target is revealed by searching for anddetecting the detection element's characteristic detection property.

One class of such probes is represented by materials known in the art as‘molecular beacons’ as for example described in U.S. Pat. No. 8,211,644.These probes are comprised of single-stranded oligonucleotides whichhave been in effect folded back onto themselves to create a residualsingle-stranded loop which acts as the probe's sensor and a short stemwhere the nucleotides adjacent the two ends are bound to each otherthrough complimentary nucleotide base pairing; thereby creating adouble-stranded region. This arrangement, which can be likened to ahairpin in which the single-stranded loop is attached to complimentarystrands of the same end of a notional double-stranded oligonucleotide,is highly strained. To the free 3′ and 5′ ends of the oligonucleotide(now adjacent to one another and at the remote end of the stem) arerespectively attached a fluorophore and a quencher. Their geometricproximity to each other then ensures that no significant fluorescenceoccurs. In use, the target binds to the single-stranded loop causingadditional strain so that when the probe is heated the stem unzipscausing distancing of the fluorophore and quencher and allowing theformer to fluoresce.

We have now developed a new sequencing method which in one embodimentinvolves generating a stream of nucleotide bases whose ordering ischaracteristic of the sequence in the analyte by progressive degradationof the analyte; and a subsequent capture of each nucleotide base in away which enables it to be detected.

WO 94/18218 discloses a genome sequencer in which an ordered stream ofsingle nucleotides is separated from an analyte and thereafter containedin a fluorescent-enhancing solid matrix where each nucleotide is excitedusing a laser and its characteristic spectroscopic emission detected.The single nucleotide transfer method used by this sequencer involvescreating a single dual-sheath of flowing immiscible liquids rather thana series of droplets. Furthermore, the sequencer described seeks todetect the single nucleotides directly rather than employing a capturesystem and fluorophore release method of the type we describe. Webelieve that this is a drawback as it will lead to signal-to-noise ratioproblems when the emission come to be detected. This will compromise theoverall sensitivity and therefore practical applicability of thesequencer itself.

Stephan et al Journal of Biotechnology 86 (2001) pp. 255-267 teaches ageneral method for counting single nucleotides generated byexonucleolytic degradation of an immobilised DNA sample labelled withfluorophores. However no information is provided about differentiatingbetween the different single nucleotide types generated.

The use of the progressive exonucleolytic degradation of polynucleotidesto generate a stream of single nucleotide bases has been disclosed inschematic form at http://www.mrc-lmb.cam.ac.uk/happy/HappyGroup/seq.htmlalthough little information about the actual methodology employed isprovided. Furthermore, WO 03/080861 describes a sequencing method inwhich a DNA analyte is sequentially degraded to an ordered stream ofsingle nucleotides by means of pyrophosphorolysis carried out in thepresence of a pyrophosphate anion labelled with an intelligent dye. Inone example the pyrophosphate anion is labelled with the dye JF-4 whichhas differing fluorescent lifetimes depending on the particularnucleotide type to which it is attached. The stream of labelled singlenucleotides is then excited by a laser and analysed spectroscopically todetermine the nature and therefore the ordering of the nucleotides. Onceagain the single nucleotides are detected directly rather than byemploying the capture system and fluorophore release method we describebelow. It is believed therefore that this method will also lead tosignal-to-noise ratio and therefore sensitivity problems.

According to the present invention there is therefore provided a methodfor determining the sequence of nucleotide bases in a polynucleotideanalyte characterised by the steps of (1) generating a stream of singlenucleotide bases from the analyte by pyrophosphorolysis; (2) producingcaptured molecules by reacting each single nucleotide base with acapture system labelled with detectable elements in an undetectablestate; (3) releasing the detectable elements from each captured moleculein a detectable state and (4) detecting the detectable elements soreleased and determining the sequence of nucleotide bases therefrom.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a microfluidic sequencing device in which a stream ofmicrodroplets at least some of which contain a single nucleotide baseare made to undergo reaction with a capture system of the first classdescribed above.

FIG. 2 shows the result of the capture of a single nucleotide base andrelease of fluorophores using a capture system over time usingradio-labelled nucleotides and gel electrophoresis. The capture of theradio-labelled nucleotides onto the j-shaped oligonucleotide occurswithin the first 2 minutes of the reaction, with ligation of the singlestranded oligonucleotide occurring over the first 30 minutes. In thisexperiment the Phusion polymerase is added at time t=30 minutes, and itcan be seen that the completed capture molecules are rapidly digested(in this case digestion occurs within 30 seconds of adding thepolymerase).

FIG. 3 shows the fluorescence measured as a function of time for thefull reaction performed in the presence (broken line) or absence (solidline) of nucleotides. In this experiment the polymerase isheat-activated at time t=20 minutes. A significant increase influorescence is observed for the reaction performed in the presence ofnucleotides, while little or no fluorescence increase is observed intheir absence.

DETAILED DESCRIPTION OF THE INVENTION

As set forth above, described herein is a method for determining thesequence of nucleotide bases in a polynucleotide analyte characterisedby the steps of (1) generating a stream of single nucleotide bases fromthe analyte by pyrophosphorolysis; (2) producing captured molecules byreacting each single nucleotide base with a capture system labelled withdetectable elements in an undetectable state; (3) releasing thedetectable elements from each captured molecule in a detectable stateand (4) detecting the detectable elements so released and determiningthe sequence of nucleotide bases therefrom.

Step (1) of the method of the present invention comprises generating astream of single nucleotide bases from the polynucleotide analyte bypyrophosphorolysis. The analyte employed in this step is suitably adouble-stranded polynucleotide comprised of many nucleotide bases. Inprincipal the length of the polynucleotide can be unlimited including upto the many millions of nucleotide bases found in a human genomefragment. The analyte itself is suitably RNA or DNA of natural originalthough the method can also be used to sequence synthetically producedRNA or

DNA or other nucleic acids made up wholly or in part of nucleotide basesthat are not commonly encountered in nature; i.e. nucleotide bases otherthan adenine, thymine, guanine, cytosine and uracil. Examples of theseinclude 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine,2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylamino-methyluridine, dihydrouridine,2-O-methylpseudouridine, 2-O-methylguanosine, inosine,N6-isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine,1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, 3-methylcytidine,5-methylcytidine, N6-methyladenosine, 7-methylguanosine,5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methoxyuridine, 5-methoxycarbonylmethyl-2-thiouridine,5-methoxycarbonylmethyluridine, 2-methylthio-N6-isopentenyladenosine,uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid,wybutoxosine, wybutosine, pseudouridine, queuosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine,2-O-methyl-5-methyluridine and 2-O-methyluridine.

In one embodiment of the invention, step (1) comprises a first sub-stepof attaching the polynucleotide analyte to a substrate. Typically, thesubstrate comprises a microfluidic surface, a micro-bead or a permeablemembrane made out of glass or a non-degradable polymer. Preferably, thesubstrate further comprises a surface adapted to receive the analyte.There are many ways in which the analyte can be attached to suchsurfaces all of which can in principle be used. For example, one methodinvolves priming a glass surface with a functionalised silane such as anepoxysilane, an aminohydrocarbylsilane or a mercaptosilane. The reactivesites so generated can then be treated with a derivative of the analytewhich has a terminal amine, succinyl or thiol group.

It is a preferable feature of step (1) that the analyte ispyrophosphorolysed to generate a stream of single nucleotide bases whoseordering corresponds to that of the analyte. This step is preferablycarried out at a temperature in the range 20 to 90° C. in the presenceof a reaction medium comprising an enzyme. Preferably it is carried outunder conditions of non-equilibrium flow so that the single nucleotidebases are continually removed from the reaction zone. Most preferably,the reaction is carried out by causing an aqueous buffered mediumcontaining the enzyme and the other typical additives to continuouslyflow over the surface to which the analyte is bound.

In one preferred embodiment, the enzyme used is one which can causeprogressive 3′-5′ pyrophosphorolytic degradation of the analyte to yielddeoxyribonucleotide triphosphates with high fidelity and at a reasonablereaction rate. Preferably this degradation rate is as fast as possibleand in one embodiment lies in the range 1 to 50, preferably 1 to 20nucleotide bases per second. Further information about thepyrophosphorolysis reaction as applied to the degradation ofpolynucleotides can be found for example in J. Biol. Chem. 244 (1969)pp. 3019-3028. The enzyme which is preferably employed in thispyrophosphorolysis reaction is suitably selected from the groupconsisting of those polymerases which show essentially neither exo- norendonuclease activity under the reaction conditions. Examples ofpolymerases which can be advantageously used include, but are notlimited to, the prokaryotic pol 1 enzymes or enzyme derivatives obtainedfrom bacteria such as Escherichia coli (e.g. Klenow fragmentpolymerase), Thermus aquaticus (e.g. Taq Pol) and Bacillusstearothermophilus, Bacillus caldovelox and Bacillus caldotenax.Suitably, the pyrophosphorolytic degradation is carried out in thepresence of a medium which further comprises pyrophosphate anion andmagnesium cations; preferably in millimolar concentrations.

In step (2) of the method of the present invention each singlenucleotide base generated in step (1) is captured by a capture systemitself comprising an oligomer of nucleotide bases. Preferably, beforethis step is carried out the aqueous medium containing the singlenucleotide bases is treated with a pyrophosphatase to hydrolyse anyresidual pyrophosphate to phosphate anion. In a first embodiment, thecapture system comprises one of a class of pairs of first and secondoligonucleotides. The first oligonucleotide in such a pair preferablycomprises (a) a first double-stranded region and (b) a secondsingle-stranded region comprised of n nucleotide bases wherein n isgreater than 1 preferably greater than 5. In one sub-class, the firstoligonucleotide can be regarded as having a molecular structure derivedfrom a notional or actual single-stranded oligonucleotide precursorwhere the double-stranded region has been created by partially foldingthe 3′ end of the precursor back on itself to generate a configurationwhich can be termed ‘j shaped’. In another sub-class, the firstoligonucleotide is generated by hybridising a third, shortersingle-stranded oligonucleotide onto the 3′ end of a longer fourthsingle-stranded oligonucleotide and then rendering the end of theresulting molecule which is double-stranded ‘blunt’ by means of aprotecting group which for example bridges the final nucleotides of thetwo strands. Typically, the total length of the first oligonucleotide isup to 150 nucleotide bases, preferably between 20 and 100 nucleotidebases. At the same time it is preferred that the integer n is between 5and 40, preferably between 10 and 30.

As regards the second oligonucleotide in the pair, this issingle-stranded and suitably has a nucleotide base sequence which iswholly or partially the compliment of that of the single-stranded regionof the first oligonucleotide starting one nucleotide base beyond the endof the double-stranded region. The length of the second oligonucleotideis not critical and can be longer or shorter than the single-strandedregion to which it can bind although it is preferably not n-1 nucleotidebases long. More preferably, the length of the second oligonucleotide ischosen so that in the captured molecule a short overhang of unpairednucleotide bases (e.g. 2 to 10 nucleotide bases) remains on one or otherof the two strands thereof. Preferably, in this class the detectableelements are located on the second oligonucleotide. Capture systems ofthis class work by attaching the single nucleotide base to thedouble-stranded end of the first oligonucleotide and hybridising thesecond oligonucleotide onto the remaining single-stranded region togenerate a captured molecule which is double-stranded apart from itsoverhang.

In a second embodiment, the capture system comprises a class of singleoligonucleotides each consisting of a single-stranded nucleotide regionthe ends of which are attached to two different double-stranded regions.In the capture systems of this class, the single-stranded nucleotideregion is comprised of one nucleotide base only making the probeextremely selective for the detection of the target i.e. thecomplimentary single nucleotide base in the droplet stream.

Turning to the double-stranded oligonucleotide region(s), it ispreferred that they are derived or derivable from two oligonucleotideprecursors, each preferably closed looped, or from a commonsingle-stranded oligonucleotide precursor by folding the latter's endsback on themselves to create two closed-loop oligonucleotide baseregions with an intermediate gap constituting the single-strandednucleotide region. In all cases the effect is the same; adjacent to theends of the single-stranded nucleotide region will be 3′ and 5′ freeends on the other strand of the oligonucleotide region to which thecorresponding 5′ and 3′ ends of the target can be attached. Thus use ofthe capture system involves a process of attaching the single-strandednucleotide region to the target single nucleotide base by joining up theavailable 3′ and 5′ ends of the capture system to generate a capturedmolecule which is double-stranded along its whole length.

Suitably, the double-stranded oligonucleotide region(s) are up to 50nucleotide base pairs long, preferably up to 45 nucleotide base pairs,more preferably in the range 5 to 40 nucleotide base pairs and mostpreferably in the range 10 to 30. Longer regions may be used but thepotential risk that access to the single-stranded nucleotide region bythe target may become restricted through entanglement. This makes thisembodiment potentially less attractive.

In this class it is preferred that the detectable elements bound to thedouble-stranded oligonucleotide region(s) are located remote from thesingle-stranded nucleotide region. Finally in one embodiment it ispreferred that at least one of the double-stranded oligonucleotideregions comprises at least one restriction enzyme recognition sitepreferably adjacent the region where the detectable elements are locatedor clustered. For these capture systems, liberation of the fluorophorescomes about by first a restriction enzyme exhibiting endonucleolyticbehaviour and making a double-stranded cut in the captured molecule atthe site mentioned above. The short fragments so created may then bedegraded further by an exonuclease into single nucleotides at least someof which will be labelled with fluorophores. Thus, when the capturedmolecule comprises multiple fluorophores this leads to the release of acascade of fluorophores which, by virtue of them now being separatedfrom each other and/or their associated quenchers, are now free tofluoresce in the normal way. Such a restriction enzyme recognition sitewill typically comprise a specific sequence of from 2 to 8 nucleotidepairs. In another preferred embodiment the restriction enzymerecognition site will be one created by binding of the single nucleotideto the single-stranded nucleotide region.

For both of the classes mentioned above, it is preferred to employ amixture of at least two different sets of capture molecules eachselective for a different complimentary nucleotide base and eachemploying a different detectable element. These may be from the same ordifferent classes. In a preferred embodiment, each set of capturemolecules will have different associated detectable elements so that,when the corresponding detection property is eventually detected, thenucleotide base can be uniquely identified. For example, when theanalyte is DNA or RNA it is most preferable to employ four differentcapture systems with each one being selective for a different nucleotidebase characteristic of these molecules.

It is a further feature of all the capture systems of the presentinvention, that they are labelled with multiple detectable elementswhich are substantially undetectable when the capture system is in anunused state. Suitably these detectable elements are ones which areadapted to be detected by an optical event. In one preferred embodiment,the detectable elements comprise fluorophores and each unused capturesystem is essentially non-fluorescing at those wavelengths where thefluorophores are designed to be detected. Thus, although a fluorophoremay exhibit general, low-level background fluorescence across a widepart of the electromagnetic spectrum, there will typically be one or asmall number of specific wavelengths or wavelength envelopes where theintensity of the fluorescence is at a maximum. It is at one or more ofthese maxima where the fluorophore is characteristically detected thatessentially no fluorescence should occur. In the context of this patent,by the term ‘essentially non-fluorescing’ or equivalent wording is meantthat the intensity of fluorescence of the total number of fluorophoresattached to the second oligonucleotide at the relevant characteristicwavelength or wavelength envelope is less than 25%; preferably less than10%; more preferably less than 1% and most preferably less than 0.1% ofthe corresponding intensity of fluorescence of an equivalent number offree fluorophores.

In principle, any method can be used to ensure that in the unused stateof the capture system the fluorophores are essentially non-fluorescing.One approach is to additionally attach quenchers in close proximity tothem. Another is based on the observation that when multiplefluorophores are attached to the capture system in close proximity toeach other they tend to quench each other sufficiently well that thecriterion described in the previous paragraph can be achieved withoutthe need for quenchers. In this context of this patent, what constitutes‘close proximity’ between fluorophores or between fluorophores andquenchers will depend on the particular fluorophores and quenchers usedand possibly the structural characteristics of the singleoligonucleotide. Consequently, it is intended that this term beconstrued with reference to the required outcome rather than anyparticular structural arrangement on the various elements of the capturesystem. However and for the purposes of providing exemplification only,it is pointed out that when adjacent fluorophores or adjacentfluorophores and quenchers are separated by a distance corresponding tothe characteristic Förster distance (typically less than 5 nm)sufficient quenching will generally be achieved.

Suitably the capture system is labelled with up to 20, for example up to10 and most preferably up to 5 fluorophores. To obtain maximumadvantage, it is preferred that the capture system is labelled with atleast 2 preferably at least 3 fluorophores. Consequently, rangesconstructed from any permutation of these maxima and minima arespecifically envisaged herein. If quenchers are employed, it is likewisepreferred that the capture system is labelled with up to 20, preferablyup to 10 and most preferably up to 5 of the same. Whilst it is envisagedthat more than one type of fluorophore can be attached to the capturesystem, for example to give it a characteristic fingerprint, it ispreferred that all the fluorophores employed in each capture system typeare the same.

As regards the fluorophores themselves, they can in principle be chosenfrom any of those conventionally used in the art including but notlimited to xanthene moieties e.g. fluorescein, rhodamine and theirderivatives such as fluorescein isothiocyanate, rhodamine B and thelike; coumarin moieties (e.g. hydroxy-, methyl- and aminocoumarin) andcyanine moieties such as Cy2, Cy3, Cy5 and Cy7. Specific examplesinclude fluorophores derived from the following commonly used dyes:Alexa dyes, cyanine dyes, Atto Tec dyes, and rhodamine dyes. Examplesalso include: Atto 633 (ATTO-TEC GmbH), Texas Red, Atto 740 (ATTO-TECGmbH), Rose Bengal, Alexa Fluor′ 750 C₅-maleimide (Invitrogen), AlexaFluor™ 532 C₂-maleimide (Invitrogen) and Rhodamine Red C₂-maleimide andRhodamine Green as well as phosphoramadite dyes such as Quasar 570.Alternatively a quantum dot or a near infra-red dye such as thosesupplied by LI-COR Biosciences can be employed. The fluorophore istypically attached to the second oligonucleotide via a nucleotide baseusing chemical methods known in the art.

Suitable quenchers are those which work by a Förster resonance energytransfer (FRET) mechanism. Examples of commercially available quencherswhich can be used in association with the above mentioned-fluorophoresinclude but are not limited to DDQ-1, Dabcyl, Eclipse, Iowa Black FQ andRQ, IR Dye-QC1, BHQ-0, BHQ-1, -2 and -3 and QSY-7 and -21.

Step (2) is suitably effected by contacting each single nucleotide basein the stream with the capture system, most preferably themulti-component capture system mentioned above, at a temperature in therange 30 to 80° C. in the presence of a two component enzyme systemcomprising a second polymerase and a ligase. In a preferred embodiment,the second polymerase is the same as that used in step (1) therebyavoiding the need to add this in the form of an extra component.

In step (3) of the method of the present invention, the detectableelements are released from the captured molecule in a detectable form byaction of an exonuclease or the exonuclease activity of a polymerase. Indoing so it is important that the fluorophores present in any of theunused sets of capture molecules are not at the same time released. Inthe case of the first class of capture system, this may be achieved forexample by using a polymerase having 3′-5′ exonuclease activity todegrade the captured molecule by virtue of its single-stranded overhangregion. Alternatively, and especially in the case of the second class ofcapture systems, this may be achieved by incorporating into the capturesystem or the captured molecule at least one restriction enzymerecognition site preferably adjacent the region where the detectableelements are located or clustered. Such a restriction enzyme recognitionsite will typically comprise a specific sequence of from 2 to 8nucleotide pairs. In a preferred embodiment of this approach, therestriction enzyme recognition site may be one created by binding of thesingle nucleotide base to the capture system.

Step (3) is also suitably carried out at a temperature in the range 30to 80° C. Suitable examples of exonucleases or polymerases which can beused in this step include Phusion, Phusion HS, Dnase I (RNase-free),Exonuclease I or III (ex E. coli), Exonuclease T, Exonuclease V(RecBCD), Lambda Exonuclease, Micrococcal Nuclease, Mung Bean Nuclease,Nuclease BAL-31, Reci_(f), T5 Exonuclease and T7 Exonuclease. The neteffect of step (3) is that the constituent nucleotides bases of thecaptured molecule will be liberated some of which will be labelled withthe characteristic detectable element. Thus, when the captured moleculecomprises multiple quenched fluorophores, this leads to a ‘cascade’ ofliberated fluorophores which, by virtue of them becoming separated fromeach other and/or their associated quenchers, are now free to fluorescein the normal way.

Thereafter, and in step (4), the detectable elements liberated from thedegraded captured molecule are detected, the particular singlenucleotide base identified and the sequence of nucleotide bases in theanalyte recovered from the data stream associated with the detection.Methods of doing this are well-known in the art; for examplefluorescence may be detected using a photodetector or an equivalentdevice tuned to the characteristic fluorescence wavelength(s) orwavelength envelope(s) of the various fluorophores. This in turn causesthe photodetector to generate an electrical signal characteristic of aparticular nucleotide base type which can be processed and thereafteranalysed.

In a particularly preferred embodiment, the method of the presentinvention is carried out wholly or partially in microdroplets. Such amethod may begin, for example, by inserting the single nucleotide basesgenerated in step (1) one-by-one into a corresponding stream of aqueousmicrodroplets in an immiscible carrier solvent such as a hydrocarbon orsilicone oil to help preserve the ordering. Advantageously, this can beeffected by directly creating the microdroplet downstream of thepyrophosphorolysis reaction zone; for example by causing the reactionmedium to emerge from a microdroplet head of suitable dimensions into aflowing stream of the solvent. Alternatively, small aliquots of thereaction medium can be sequentially injected into a stream ofpre-existing aqueous microdroplets suspended in the solvent. If thislatter approach is adopted, each microdroplet may suitably contain thevarious components of the capture system and the enzymes and any otherreagents (e.g. buffer) required to effect steps (2) and (3). Finally,the microdroplets created in the former embodiment can be caused tocoalesce subsequently with a stream of such pre-exiting microdroplets toachieve a similar outcome. In this embodiment, step (4) then preferablyinvolves interrogating each droplet in turn to identify the detectableelements liberated and hence the nature of the nucleotide base itcontains.

To avoid the risk that a given microdroplet contains more than onesingle nucleotide base, it is preferred to release the single nucleotidebases in step (1) at a rate such that each filled microdroplet isseparated by from 1 to 20 preferably 2 to 10 empty ones. Thereafter thestream of filled and unfilled microdroplets in the solvent is caused toflow along a flow path, suitably a microfluidic flow path, at a rate andin a manner such that the microdroplets are maintained in a discretestate and do not have the opportunity to coalesce with each other.Suitably the microdroplets employed have a diameter less than 100microns, preferably less than 50 microns, more preferably less than 20microns and even more preferably less than 15 microns. Most preferablyof all their diameters are in the range 2 to 20 microns. In oneembodiment, the microdroplet flow rate through the whole system is inthe range 50 to 3000 droplets per second preferably 100 to 2000.

The method described above can be used to advantage in a sequencingdevice and such devices are envisaged as being within the scope of theinvention.

The present invention will now be illustrated with reference to thefollowing examples.

Preparation and Use of a Capture System

The following experiment illustrates the capture of a single nucleotidebase and release of fluorophores using a capture system wherein thefirst oligonucleotide is j-shaped and the second is single-stranded.

A sample of a j-shaped oligonucleotide as described above is prepared byfolding a 75 nucleotide base, single-stranded oligonucleotide having thefollowing sequence:gtaggtcctggcacagaaaaaaggagGcagtgatgttccatgactgatttttttttcagtcatggaacatcact*gwherein g, t, c, and a represent the conventional notation for thenucleotide bases of DNA and * represents the presence of aphosphorothioate linkage. Folding is carried out by heating an aqueoussolution of this oligonucleotide to 95° C. and then cooling it slowlyback to room temperature at a rate of 10 minutes per ° C. The j-shapedmolecule so obtained comprises a residual single-strandedoligonucleotide region (gtaggtcctggcacagaaaaaaggag) attached to a singlenucleotide base which is the site of capture (capitalised in theabove-mentioned sequence).

A corresponding single-stranded oligonucleotide is also prepared, havingthe following sequence:

̂ctccTTXTTtctgtgccagawherein ̂ represents a 5′ phosphate group, a capitalised T represents athymine base labelled with Alexa Fluor 488 dye via an azide linker, andan X represents a thymine base labelled with a BHQ-0 quencher.

Separate capture and nucleotide base mixtures are then prepared. Thecapture mixture has a composition corresponding to that derived from thefollowing formulation:

2.5 ul 10× BufferII

5 ul 10× Taq Ligase buffer (NEB)2.5 ul 100 nM of the j-shaped molecules mentioned above5 ul 100 nM of the single-stranded oligonucleotide mentioned above

2 ul Thermostable Inorganic Pyrophosphatase (NEB) 5 ul Taq Ligase (NEB)1 ul 25 mM MnSO4

water to 25 ulwhilst the nucleotide base mixture, whose composition is designed tomimic the material, obtained from the pyrophosphorolysis step,corresponds to that derived from the formulation:2.5 ul 10 BufferII (supplied with Amplitaq; magnesium-free)

1.5 ul MgCl2 25 mM

2.5 ul 10 nM of deoxycytidine triphosphate (dCTP)

2 ul Amplitaq (5 U/ul)

2.5 ul 10 mM sodium pyrophosphatewater to 25 ul.Capture of the dCTP is then effected by mixing together equal volumes ofthese two mixtures and incubating the resulting product at 50° C. Thisis typically complete in 30 minutes. At the end of this time a sample ofthe mixture (50 ul) is treated with 1 ul HotStart Phusion DNA polymerase(NEB) and activated at 98° C.×20 s so that exonucleolytic degradation ofthe completed capture molecules can occur. Degradation is typicallycomplete within 30 minutes, and the released fluorophores can bedetected by illuminating the sample at or close to the peak absorptionwavelength (496 nm), and detecting the resulting fluorescence at thecharacteristic emission wavelength (519 nm).

FIG. 2 shows the result of this reaction over time using radio-labellednucleotides and gel electrophoresis. The capture of the radio-labellednucleotides onto the j-shaped oligonucleotide occurs within the first 2minutes of the reaction, with ligation of the single strandedoligonucleotide occurring over the first 30 minutes. In this experimentthe Phusion polymerase is added at time t=30 minutes, and it can be seenthat the completed capture molecules are rapidly digested (in this casedigestion occurs within 30 seconds of adding the polymerase).

FIG. 3 shows the fluorescence measured as a function of time for thefull reaction performed in the presence (broken line) or absence (solidline) of nucleotides. In this experiment the polymerase isheat-activated at time t=20 minutes. A significant increase influorescence is observed for the reaction performed in the presence ofnucleotides, while little or no fluorescence increase is observed intheir absence.

Droplet Microfluidic Method Using the Capture System

FIG. 1 illustrates a microfluidic sequencing device in which a stream ofmicrodroplets at least some of which contain a single nucleotide baseare made to undergo reaction with a capture system of the first classdescribed above.

An aqueous medium 1 comprising a stream of discrete deoxyribonucleotidetriphosphates obtained by the progressive pyrophosphorolysis of a 100nucleotide base polynucleotide analyte derived from human DNA is causedto flow through a ten micron diameter microfluidic tube fabricated fromPDMS polymer. The pyrophosphorolysis reaction itself is carried out bypassing a stream of an aqueous, buffered (pH 8) reaction medium at 72°C., comprising Taq Pol and a 2 millimoles per litre concentration ofeach of sodium pyrophosphate and magnesium chloride, over a glass microbead onto which the analyte has been previously attached by means of asuccinyl bridge. The order of the single nucleotide bases in stream 1,which is downstream of the micro bead, corresponds to the sequence ofthe analyte. 1 emerges from a droplet head 2 into a first chamber 3where it is contacted with one or more streams of immiscible lightsilicone oil 4. The velocities of these streams are chosen to avoidturbulent mixing and to create in 3 aqueous spherical droplets 5suspended in the oil each having a diameter of approximately eightmicrons. Typically, the rate of pyrophosphorolysis and/or the rate offlow of 1 are adjusted so that between adjacent filled droplets thereare 10 empty ones. A stream of 5 is then carried forward along a secondmicrofluidic tube of the same diameter at a rate of 1000 droplets persecond to a second chamber 6 into which a second stream of five micronaqueous spherical droplets 7 is also fed by means of a second droplethead 8. Droplets 5 and 7 are caused to coalesce in a sequential fashionto form enlarged aqueous droplets 9 approximately nine microns indiameter. Each of 7 contains pyrophosphatase to destroy any residualpyrophosphate anion present in each of 5.

A stream of 9 is then carried forward at the same rate via microfluidictubing into a third chamber 10 where these droplets are contacted with athird stream of five micron aqueous spherical droplets 11 also fedthereto through a corresponding droplet head 12. The time taken for eachof 9 to move between chambers 6 and 10 is c.2 minutes.

Droplets 9 and 11 are then caused to coalesce in 10 to produce droplets13 (approximately ten microns in diameter). Each of 11 contains amesophilic ligase and a capture system comprising four pairs of j-shapedfirst oligonucleotides and four corresponding second single-strandedoligonucleotides. In this example, each j-shaped first oligonucleotideis 60 nucleotide bases long and is prepared by folding a 60 nucleotidebase single-stranded oligonucleotide precursor about the 45^(th)nucleotide base from the 5′ end to generate a 3 nucleotide base singlestranded loop, a 12 nucleotide base pair double-stranded region and a 33nucleotide base single-stranded region. Each of these four firstoligonucleotides has a different 33^(rd) base (measured from thesingle-stranded end) characteristic of the four characteristicnucleotide base types of DNA (i.e. A, T, G and C). The four differentsecond oligonucleotides are each 28 nucleotide bases long and havesequences which are complimentary to that part of the single-strandedregion defined by the 4^(th) and 32^(nd) nucleotide bases of their firstoligonucleotide pair. The four different second oligonucleotide typesare labelled respectively with the fluorophores Quasar 570, Fluorescein,Texas Red and Cy-5 (five fluorophores moieties per secondoligonucleotide). In each case fluorescence is quenched by the inclusionof one quencher moiety on each second oligonucleotide (BHQ-2 for Quasar570 and Texas Red, BHQ-0 for Fluorescein and BHQ-3 for cyanine-5).

A stream of 13 is next carried forward at the same rate via microfluidictubing into a fourth chamber 14 where it is caused to coalesce with afourth stream of five micron aqueous spherical droplets 15 also fedthereto through a droplet head 16. The time taken for each of 9 to movebetween the two chambers is 30 minutes in which time the singlenucleotide base is captured by its capture system pair and the capturedmolecule formed. Each of 15 contains Phusion exonuclease to degrade thecapture molecule and release the relevant fluorophores in detectableform. A stream of the coalesced microdroplets 17 is then taken forwardto a container 18 in which their progress is tracked until they reachone of array of sites 19a where they are held 19b until such time asthey are analysed.

After 2 hours each droplet held in the array is illuminated in turn andin the correct order with one or more high intensity light sources, forexample one or more lasers emitting coherent light at the relevantfrequencies of the fluorophores and the fluorescence so generateddetected by a photodetector operating at those wavelengthscharacteristic of the four fluorophore types. From the informationreceived the single nucleotide base is identified in each droplet andnil responses from empty droplets rejected. The results are thenprocessed by a computer programmed to recreate the original nucleotidebase sequence of the analyte. If so desired, multiple cycles ofillumination and detection can be performed across the array of dropletsat various intervals which can be averaged to improve the single tonoise ratio and therefore the reliability of the results.

1-24. (canceled)
 25. A method for identifying the nucleotide base in asingle nucleoside triphosphate molecule, the method comprising the stepsof: (a) producing a captured molecule by reacting the nucleosidetriphosphate molecule with a capture system comprising anoligonucleotide selective for the nucleotide base and labelled with atleast one characteristic fluorophore in an undetectable state; (b)releasing the fluorophore(s) from the captured molecule in a detectablestate; (c) detecting characteristic fluorescence from the fluorophore(s)so released; and (d) inferring therefrom the identity of the nucleosidetriphosphate molecule.
 26. The method of claim 25, wherein the capturesystem is comprised of two oligonucleotide components; (a) a firstoligonucleotide comprising a double-stranded region and asingle-stranded nucleotide region and (b) a second single-strandedoligonucleotide whose nucleotide base sequence is at least partiallycomplimentary to that of the single-stranded region of the firstoligonucleotide.
 27. The method of claim 25, wherein the capture systemcomprises a single oligonucleotide component comprising asingle-stranded nucleotide region complementary to the nucleotide baseof the nucleoside triphosphate molecule the 3′ and 5′ ends of which areattached to two different double-stranded oligonucleotide regions. 28.The method of claim 26, wherein the first oligonucleotide is j-shaped.29. The method of claim 26, wherein the total length of the firstoligonucleotide is from 20 to 100 nucleotides.
 30. The method of claim25, wherein the fluorophores attached to the oligonucleotide arerendered undetectable by the presence of at least one quencher.
 31. Themethod of claim 25, carried out in the presence of up to four differentcapture systems each selective for a different one of the fournucleotide base types characteristic of DNA or RNA.
 32. The method ofclaim 31, wherein each capture system is comprised of a differentoligonucleotide labelled with a different fluorophore fluorescing at adifferent wavelength.
 33. The method of claim 32, wherein eacholigonucleotide is labelled with a quencher.
 34. The method of claim 25,wherein the fluorophores are released from the captured molecule in adetectable state using an exonuclease or the exonuclease activity of apolymerase.
 35. The method of claim 25, wherein the captured moleculefurther comprises at least one restriction enzyme recognition site,wherein the captured molecule undergoes endonucleolysis before thefluorophores are released in a detectable state.
 36. The method of claim35, wherein the restriction enzyme recognition site is created byreacting the single nucleoside triphosphate molecule with the capturesystem.
 37. The method of claim 35, wherein the fluorophores arereleased from the captured molecule in a detectable state using anexonuclease or the exonuclease activity of a polymerase.
 38. The methodof claim 36, wherein the fluorophores are released from the capturedmolecule in a detectable state using an exonuclease or the exonucleaseactivity of a polymerase.
 39. The method of claim 25, wherein thereaction between the nucleoside triphosphate molecule and the capturesystem is carried out in the presence of a polymerase and a ligase. 40.The method of claim 25, wherein the fluorophores are released from thecaptured molecule in a detectable state in the form of labelled singlenucleotides.
 41. The method of claim 25, wherein at least one of thesteps is carried out in a microdroplet.
 42. The method of claim 25,wherein the single nucleoside triphosphate molecule is produced bypyrophosphorolysis of a polynucleotide analyte.
 43. The method of claim42, wherein the polynucleotide analyte is derived fromnaturally-occurring DNA or RNA.
 44. A method for identifying thenucleotide base associated with a given single nucleoside triphosphatemolecule, the method comprising the steps of: (a) producing a capturedmolecule by reacting the nucleoside triphosphate molecule with a capturesystem comprising an oligonucleotide selective for the nucleotide baseand labelled with at least one characteristic fluorophore in anundetectable state; (b) releasing single nucleotides labelled with thefluorophore(s) from the captured molecule by exonucleolysis; (c)detecting characteristic fluorescence from the fluorophores so released;and (d) inferring therefrom the identity of the nucleoside triphosphatemolecule.
 45. The method of claim 44, wherein the captured moleculefurther comprises at least one restriction enzyme recognition site,wherein the captured molecule undergoes endonucleolysis before thesingle nucleotides are released by exonucleolysis.
 46. The method ofclaim 45, wherein the restriction enzyme recognition site is created byreacting the single nucleoside triphosphate molecule with the capturesystem.